title: Improvement of LATE-PCR to allow single-cell analysis by pyrosequencing.
abstract: Nucleic acid analysis in a single cell is very important, but the extremely small amount of template in a single cell requires a detection method more sensitive than the conventional method. In this paper, we describe a novel assay allowing a single cell genotyping by coupling improved linear-after-the-exponential-PCR (imLATE-PCR) on a modified glass slide with highly sensitive pyrosequencing. Due to the significantly increased yield of ssDNA in imLATE-PCR amplicons, it is possible to employ pyrosequencing to sequence the products from 1 μL chip PCR which directly used a single cell as the starting material. As a proof-of-concept, the 1555A>G mutation (related to inherited deafness) on mitochondrial DNA and the SNP 2731C>T of the BRCA1 gene on genomic DNA from a single cell were successfully detected, indicating that our single-cell-pyrosequencing method has high sensitivity, simple operation and is low cost. The approach has promise to be of efficient usage in the fields of diagnosis of genetic disease from a single cell, for example, preimplantation genetic diagnosis (PGD).
authors.affiliation: Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Qinxin
authors.initials: Q
authors.lastname: Song
authors.forename: Huiyong
authors.initials: H
authors.lastname: Yang
authors.forename: Bingjie
authors.initials: B
authors.lastname: Zou
authors.forename: Tomoharu
authors.initials: T
authors.lastname: Kajiyama
authors.forename: Hideki
authors.initials: H
authors.lastname: Kambara
authors.forename: Guohua
authors.initials: G
authors.lastname: Zhou
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics; D059018:DNA Contamination; D004272:DNA, Mitochondrial / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D016014:Linear Models; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D019313:BRCA1 Protein; D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded